We examined the effect of insulin supplementation and hypoxic culture (2% vs. 20% oxygen tension) on collagen deposition and mechanical properties of fibrin-based tubular tissue constructs seeded with neonatal human dermal fibroblasts. The results presented here demonstrate that constructs cultured under hypoxic conditions with insulin supplementation increased in collagen density by approximately five-fold and both the ultimate tensile strength (UTS) and modulus by more than three-fold compared with normoxic (20% oxygen tension), noninsulin supplemented controls. In addition, collagen deposited on a per-cell basis increased by approximately four-fold. Interaction was demonstrated for hypoxia and insulin in combination in terms of UTS and collagen production on a per-cell basis. This interaction resulted from two distinct processes involved in collagen fibril formation. Western blot analysis showed that insulin supplementation alone increased Akt phosphorylation and the combined treatment increased collagen prolyl-4-hydroxylase. These molecules are distinct regulators of collagen deposition, having an impact at both the transcriptional and posttranslational modification stages of collagen fibril formation that, in turn, increase collagen density in the tissue constructs. These findings highlight the potential of utilizing insulin supplementation and hypoxic culture in combination to increase the mechanical strength and stiffness of fibrin-based engineered tissues.